Melanoma associated peptide analogues and vaccines against melanoma

ABSTRACT

The present invention is concerned with cancer treatment and diagnosis, especially with melanoma associated peptide analogues with improved immunogenicity, epitopes thereof; vaccines against melanoma, tumor infiltrating T lymphocytes recognizing the antigen and diagnostics for the detection of melanoma and for the monitoring of vaccination. The peptides according to the invention can be exploited to elicit native epitope-reactive Cm. Usage of the peptides with improved immunogenicity may contribute to the development of CTL-epitope based vaccines in viral disease and cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/214,836filed Oct. 4, 1999, which was the National Stage of InternationalApplication No. PCT/EP97/03712 filed Jul. 8, 1997 and having a prioritydate of Jul. 11, 1996. The disclosure of each of these relatedapplications is incorporated herein in their entireties.

BACKGROUND OF THE INVENTION

The present invention is concerned with cancer treatment and diagnosis,especially with melanoma associated peptide analogues, epitopes thereof,vaccines against and diagnostics for the detection of melanoma and forthe monitoring of vaccination.

During the stepwise changes from normal to tumor tissue,tumor-associated antigens appear. The characteristics oftumor-associated antigens are very much dependent on the origin of thetumor carrying them. The existence of antigens associated with animaltumors was documented in the last century, and the antigenic characterof human cancers has been well established, primarily through recentstudies with monoclonal antibodies.

Attempts to isolate and chemically characterize these antigens haveencountered serious difficulties, many having to do with a lack ofreagents suitable for precipitation of the antigen-bearing moleculesfrom a solution.

Like many other stimuli, the tumor-associated antigens activate not onebut a whole set of defense mechanisms—both specific and unspecific,humoral and cellular. The dominant role in in vivo resistance to tumorgrowth is played by T lymphocytes. These cells recognizetumor-associated antigens presented to them by antigen presenting cells(APCs), and will be activated by this recognition, and upon activationand differentiation, attack and kill the tumor cells.

Cytotoxic T lymphocytes (CTL) recognize short peptide fragments of 9-11amino acids in length, which are presented in the antigen-binding grooveof Major Histocompatibility Complex (MHC) class 1 molecules (Townsend etal., 1986, Cell 44.959; Bjorkman et al., 1987, Nature 329:512). Thesepeptides are usually derived from intracellular protein pools andassociate in the lumen of the endoplasmic reticulum with MHC class Iheavy chain and 132-microglobulin molecules, followed by transportationof the MHC-peptide complex to the cell surface. Despite the presence ofmany putative antigenic peptides within the same antigen, only a fewpeptides are selected for recognition by CTL.

MHC Class I/II antigens are often down regulated in solid tumors. Thismay affect all class I/II antigens, or only part of them. Viral andcellular peptides that can sensitize appropriate target cells forcytotoxic T lymphocyte mediated lysis may fail to do so when produced incells with a low level of expression of MHC class I antigen. Cytotoxicsensitivity may be induced, at least in some cases by raising the levelof MHC class I/II antigen expression by interferon γ and tumor necrosisfactor α.

The MHC class I binding-affinity of an epitope is an important parameterdetermining the immunogenicity of the peptide-MHC complex. Analysis ofHuman histocompatibility antigen (HLA-A *0201)-restricted epitopesrecognized by anti-viral CTL demonstrated that several peptides bind toHLA-A *0201 with high affinity. Furthermore, immunogenicity analysis ofmotif containing potential epitopes using HLA-A *0201 transgenic micerevealed that a threshold MHC class I affinity was required for apeptide in order to elicit a CTL response (Ressing et al., 1995, J.Immunol. 154:5934; Sette et al., 1994, J. Immunol. 153:5586). Inaddition to the MHC class I-binding affinity, stability of peptide-MHCcomplexes at the cell surface contributes to the immunogenicity of a CTLepitope. Consequently, MHC class I binding-affinity and stability ofpeptide-MHC complexes are important criteria in the selection ofspecific peptide determinants for development of CTL-epitope basedtherapeutic vaccines.

Recently, a number of antigens have been identified as target antigensfor anti-melanoma CTL. Using a genetic approach, the tumor specificantigens MAGE-1 and -3, as well as the melanocyte-lineage specificantigen tyrosinase, were identified (van der Bruggen et al., 1991,Science 254:1643; Gaugler et al., 1994, J. Exp. Med. 179:921; Brichardet al., 1993, J. Exp. Med. 178:489).

In the co-owned and co-pending patent-application (EP 0 668 350), thegp100 melanocyte-specific protein was identified as a target antigen formelanoma tumor infiltrating lymphocytes.

Recently, two other melanocyte differentiation antigens, Melan-A/MART-1and gp75, were identified as target antigens for anti-melanoma CTL(Coulie et al., 1994, J. Exp. Med. 180:35; Kawakami et al., 1994, Proc.Natl. Acad. Sci. USA. 91:3515; Wang et al., 1995, (vol 181, pg 799,1995). J. Exp. Med. 181:1261. 10-12). Eight HLA-A *0201 restrictedepitopes derived from these antigens have now been characterized,displaying varying affinities for HLA-A *0201 (Wolfel et al., 1994, Eur.J. Immunol. 24:759; Cox et al, 1994, Science 264:716; Kawakami et al.1995. J. Immunol. 154:3961; Bakker et al., 1995, Int. J. Cancer 62:97;Kawakami et al., 1994, J. Exp. Med. 180:347; Castelli et al., 1995, J.Exp. Med. 181:363).

DISCLOSURE OF THE INVENTION

In an attempt to improve the immunogenicity of two HLA-A *0201 presentedepitopes derived from the melanocyte differentiation antigens gp 100 andMelan-A/MART-1, amino acid substitutions within the epitopes to improveHLA-A *0201-binding affinity were performed.

Surprisingly, it was found that these epitope-analogues have an improvedimmunogenicity in view of the original epitope. Furthermore, in thepresent invention it is demonstrated that the epitope-analogues allowthe induction of peptide-specific CTL displaying cross-reactivity withtarget cells endogenously processing and presenting the native epitope.

Usage of these epitope-analogues according to the present invention withimproved immunogenicity may contribute to the development of CTL-epitopebased vaccines in chronic viral disease and cancer.

In more detail, since MHC class I-affinity and peptide-MHCcomplex-stability are important parameters determining theimmunogenicity of an MHC class I presented epitope, the possibility toimprove the capacity of two melanocyte differentiation antigen-derivedepitopes to bind to HLA-A *0201 without affecting interactions with theT-cell receptor (TCR) is explored. Detailed analysis of theMelan-AIMART-1 27-35 and gp100 154-162 epitopes using alaninesubstitutions revealed that amino acids at positions 4 to 7(Melan-A/MART-1 27-35) or 5 to 7 (gp100 154-162) are critical residuesfor TCR recognition. These data are in line with X-ray crystallographystudies of the HLA-A *0201 molecule (Saper et al., 1991, J. Mol. Biol.219:277; Latron et al., 1992, Science 257:964); implying a role for themore permissive residues at position 4 and 5 of the peptide orientedtowards the outside of the MHC molecule, as prominent TCR contact sites.It is demonstrated that for HLA-A *0201 the amino acids at positions 6and 7 of the Melan-A/MART-1 27-35 and gp100 154-162 epitopes do not onlyinteract with secondary pockets in the MHC peptide-binding cleft, butthat they are also critical residues for TCR interaction (Ruppert etal., 1993, Cell 74:929; Madden et al., 1993, Cell 75:693).

Surprisingly, the alanine substitution at position 8 in the gp100154-162 epitope, KTWGQYWAV (SEQ ID NO: 1), resulted in a peptide thatdisplayed increased HLA-A *0201 affinity. Moreover, thisepitope-analogue was recognized by gp100-reactive CTL at tenfold lowerconcentrations compared to the native epitope. These data demonstratethat amino acid substitutions at a non-anchor position can result inincreased MHC class I affinity and T cell recognition.

By N-terminal anchor replacements with V, L, M or I towards the HLA-A*0201 binding-motifs were set out to identify epitope-analogues for bothepitopes with improved affinity for HLA-A *0201 that were stillrecognized by wild type epitope-reactive CTL. For the Melan-A/MART-1epitope, epitope-analogues were obtained with comparable (M) or improved(V, L and I) affinity for HLA-A *0201. However, all N-terminal anchorreplacements resulted in decreased T cell reactivity. Apparently, incase of this epitope, the N-terminal anchoring residue affects thepositioning of the side chains in the center of the peptide, therebyabrogating TCR interactions. Recently, a similar observation has beendescribed involving an HLA-B*3501 restricted epitope of the influenza Amatrix protein (Dong et al., 1996, Eur. J. Immunol. 26:335).Substitution of a serine residue at position 2 of the peptide for themore common HLA-B*3501 N-terminal anchor proline, considerably enhancedbinding to HLA-B*3501, but the epitope-analogue was not recognized byCTL reactive with the native epitope. Moreover, this peptide behaved asa peptide-antagonist as was demonstrated for T cell recognition of bothMEC class II and class I-presented peptides (Dong et al., 1996, Eur. J.Immunol. 26:335; De Magistris et al., 1992, Cell 68:625; Klenerman etal., 1994, Nature 369:403). These findings illustrate that anchorresidue substitutions not only affect MHC class I binding, but in somecases they may also result in a conformational change of the peptide-MHCcomplex, leading to an altered interaction with the TCR.

However, in case of the gp100 154-162 epitope, in addition to thealanine substituted analogue KTWGQYWAV (SEQ ID NO: 1), three anchorsubstituted epitope-analogues KVWGQYWQV (SEQ ID NO: 2), KLWGQYWQV (SEQID NO: 3), and KIWGQYWQV (SEQ ID NO: 4), with improvedHLA-A*0201-affinity that were recognized by anti-gp100 CTL at tenfoldlower concentrations compared to the wild type epitope were obtained. Invivo immunization experiments using HLA-A*0201/K^(b) transgenic micedemonstrated that these epitope-analogues were immunogenic, resulting inthe induction of murine CTL reactive with both the epitope-analogues andthe native epitope. The immunogenicity of the epitope-analogues wasexpected since the peptide-MHC complex stability of both theepitope-analogues and the native epitope was comparably high.

In vitro CTL induction experiments using donor derived PBL demonstratedthat epitope-analogue specific CTL could be obtained displayingcross-reactivity with tumor cells endogenously presenting the wild typeepitope. In addition to T lymphocytes reactive with the wild typeepitope, the T cell repertoire of healthy donors apparently alsocontains T cells reactive with the gp100 154-162 epitope-analogues.Analysis of TCR usage of cloned CTL reactive with the different gp100154-162 epitope-analogues and with, wild type gp100 154-162 will beinformative of the spectrum of the T cell repertoire that can be used toinduce CTL reactivity towards the wild type epitope. With respect toimmunotherapy of cancer, activation of multiple specificities in the Tcell repertoire against an antigenic tumor epitope usingepitope-analogues may increase the possibility of a patient to mount asuccessful anti-tumor response upon immunization. In addition, modifiedepitopes might still elicit immune responses if tolerance against thewild-type epitope is observed.

Employment of “improved” epitopes in immunotherapy protocols increasesthe amount of peptide-MHC complexes at the cell surface of antigenpresenting cells in vivo, and will result in enhanced priming ofantigen-specific CTL. Apart from their potential in cancerimmunotherapy, usage of epitope-analogues with improved immunogenicitymay contribute to the development of CTL-epitope based vaccines inchronic viral disease.

Therefore, the present invention includes peptides, immunogenic withlymphocytes directed against metastatic melanomas, characterized in thatit comprises at least part of the amino-acid sequence of SEQ ID NO: 9wherein the amino-acid at position 2 or 8 is substituted.

A preferred embodiment of the present invention are peptides, wherein atposition 2 Threonine is substituted by Isoleucine, Leucine or Valine.

Another preferred embodiment of the present invention are peptides,wherein at position 8 Glutamine is substituted by Alanine.

A specific preferred embodiment of the present invention are peptides,characterized in that it comprises the amino-acid sequence of any of SEQID NOS: 1-4 or 32-34.

The term “peptide” refers to a molecular chain of amino acids, does notrefer to a specific length of the product and if required can bemodified in vivo or in vitro, for example by manosylation,glycosylation, amidation, carboxylation or phosphorylation: thus interalia polypeptides, oligopeptides and proteins are included within thedefinition of peptide. In addition, peptides can be part of a (chimeric)protein or can be (part of) an RNA or DNA sequence encoding the peptideor protein.

Of course, functional derivatives as well as fragments of the peptideaccording to the invention are also included in the present invention.Functional derivatives are meant to include peptides which differ in oneor more amino acids in the overall sequence, which have deletions,substitutions, inversions or additions. Amino acid substitutions whichcan be expected not to essentially alter biological and immunologicalactivities have been described. Amino acid replacements between relatedamino acids or replacements which have occurred frequently in evolutionare, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof,M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found.,Washington D.C., 1978, vol. 5, suppl. 3). Based on this information,Lipman and Pearson developed a method for rapid and sensitive proteincomparison (Science 227, 1435-1441, 1985) and determining the functionalsimilarity between homologous polypeptides.

Furthermore, as functional derivatives of these peptides are also meantto include other peptide-analogues derived from gp100 (or Melan) thatare able to induce target cell lysis by tumor infiltrating lymphocytes.

In addition, with functional derivatives of these peptides are alsomeant addition salts of the peptides, amides of the peptides andspecifically the C-terminal amides, esters and specifically theC-terminal esters and N-acyl derivatives specifically N-terminal acylderivatives and N-acetyl derivatives.

The peptides according to the invention can be produced synthetically,by recombinant DNA technology or by viruses, if the amino acid sequenceof the peptide is encoded by a DNA sequence which is part of the virusDNA. Methods for producing synthetic peptides are well known in the art.

The organic chemical methods for peptide synthesis are considered toinclude the coupling of the required amino acids by means of acondensation reaction, either in homogenous phase or with the aid of aso-called solid phase. The condensation reaction can be carried out asfollows:

condensation of a compound (amino acid, peptide) with a free carboxylgroup and protected other reactive groups with a compound (amino acid,peptide) with a free amino group and protected other reactive groups, inthe presence of a condensation agent;

condensation of a compound (amino acid, peptide) with an activatedcarboxyl group and free or protected other reaction groups with acompound (amino acid, peptide) with a free amino group and free orprotected other reactive groups.

Activation of the carboxyl group can take place, inter alia, byconverting the carboxyl group to an acid halide, azide, anhydride,imidazolide or an activated ester, such as the N-hydroxy-succinimide,N-hydroxy-benzotriazole or p-nitrophenyl ester.

The most common methods for the above condensation reactions are: thecarbodiimide method, the azide method, the mixed anhydride method andthe method using activated esters, such as described in The Peptides,Analysis, Synthesis, Biology Vol. 1-3 (Ed. Gross, E. and Meienhofer, J.)1979, 1980, 1981 (Academic Press, Inc.).

Production of peptides by recombinant DNA techniques is a general methodwhich is known, but which has a lot of possibilities all leading tosomewhat different results. The polypeptide to be expressed is coded forby a DNA sequence or more accurately by a nucleic acid sequence.

Also part of the invention is the nucleic acid sequence comprising thesequence encoding the peptides according to the present invention.

Preferably, the sequence encoding the peptides according to the presentinvention are the sequences shown in SEQ ID NOS: 1-4 and 32-34.

As is well known in the art, the degeneracy of the genetic code permitssubstitution of bases in a codon to result in another codon still codingfor the same amino acid, e.g., the codon for the amino acid glutamicacid is both GAT and GAA. Consequently, it is clear that for theexpression of a polypeptide with an amino acid sequence as shown in SEQID NO: 1-4, 9 or 32-34 use can be made of a derivate nucleic acidsequence with such an alternative codon composition thereby differentnucleic acid sequences can be found.

“Nucleotide sequence” as used herein refers to a polymeric form ofnucleotides of any length, both to ribonucleic acid (RNA) sequences andto deoxyribonucleic acid (DNA) sequences. In principle, this term refersto the primary structure of the molecule. Thus, this term includesdouble and single stranded DNA, as well as double and single strandedRNA, and modifications thereof.

A further part of the invention are peptides, which are immunogenicfragments of the peptide-analogues.

Immunogenic fragments are fragments which still have the ability toinduce an immunogenic response, i.e., that it is either possible toevoke antibodies recognizing the fragments specifically, or that it ispossible to find T lymphocytes which have been activated by thefragments. Another possibility is a DNA vaccine.

As has been said above, it has been known that the immunogenic action oftumor associated antigens is often elicited through a T cell activatingmechanism (Townsend et al., 1989, H., Ann. Rev. Immunol. 1601-624).Cytotoxic T lymphocytes (CTLs) recognizing melanoma cells in a T-cellreceptor (TCR)-dependent and MHC-restricted manner have been isolatedfrom tumor-bearing patients (Knuth et al., 1992, Cancer surveys, 39-52).It has been shown that a peptide derived from tyrosinase, anothermelanocyte-specific antigen, is recognized by a CTL clone (Brichard etal., 1993, J. Exp. Med., 178, 489-495).

It is known that the activation of T cells through the MHC moleculenecessitates processing of the antigen of which short pieces (forexample 8-12 mers) are presented to the T lymphocyte.

Preferably, the peptides according to the present invention are flankedby non-related sequences, i.e., sequences with which they are notconnected in nature, because it has been found that such flankingenhances the immunogenic properties of these peptides, probably througha better processing and presentation by APCs.

Another part of the invention is formed by nucleotide sequencescomprising the nucleotide sequences coding for the above mentionedpeptides or an array of peptides.

Next to the use of these sequences for the production of the peptideswith recombinant DNA techniques, which will be exemplified further, thesequence information disclosed in the sequence listings for the peptidesaccording to the present invention can be used for diagnostic purposes.

From these sequences primers can be derived as basis for a diagnostictest to detect gp100 or gp100-like proteins by a nucleic acidamplification technique for instance the polymerase chain reaction (PCR)or the nucleic acid sequence based amplification (NASBA) as described inU.S. Pat. No. 4,683,202 and EP 329,822, respectively.

These nucleotide sequences can be used for the production of thepeptides according to the present invention with recombinant DNAtechniques. For this, the nucleotide sequence must be comprised in acloning vehicle which can be used to transform or transfect a suitablehost cell.

A wide variety of host cell and cloning vehicle combinations may beusefully employed in cloning the nucleic acid sequence. For example,useful cloning vehicles may include chromosomal, non-chromosomal andsynthetic DNA sequences such as various known bacterial plasmids, andwider host range plasmids such as pBR 322, the various pUC, pGEM andpBluescript plasmids, bacteriophages, e.g. lambda-gt-Wes, Charon 28 andthe M13 derived phages and vectors derived from combinations of plasmidsand phage or virus DNA, such as SV40, adenovirus or polyoma virus DNA(Rodriquez et al., 1988, ed. Vectors, Butterworths; Lenstra et al.,1990, Arch. Vivol., 110, 1-24).

Useful hosts may include bacterial hosts, yeasts and other fungi, plantor animal hosts, such as Chinese Hamster Ovary (CHO) cells, melanomacells, dendritic cells, monkey cells and other hosts.

Vehicles for use in expression of the peptides may further comprisecontrol sequences operably linked to the nucleic acid sequence codingfor the peptide. Such control sequences generally comprise a promotersequence and sequences which regulate and/or enhance expression levels.Furthermore, an origin of replication and/or a dominant selection markerare often present in such vehicles. Of course, control and othersequences can vary depending on the host cell selected.

Techniques for transforming or transfecting host cells are quite knownin the art (for instance, Maniatis et al., 1982/1989, Molecular cloning:A laboratory Manual, Cold Spring Harbor Lab.).

It is extremely practical if, next to the information for the peptide,also the host cell is co-transformed or co-transfected with a vectorwhich carries the information for an MHC molecule to which said peptideis known to bind. Preferably, the MHC molecule is HLA-A2.1, HLA-A1 orHLA-A3.1, or any other HLA allele which is known to be present inmelanoma patients. HLA-A2.1 is especially preferred because it has beenestablished (Anichini et al., 1993, J. Exp. Med., 177, 989-998) thatmelanoma cells carry antigens recognized by HLA-A2.1 restrictedcytotoxic T cell clones from melanoma patients.

Host cells especially suited for the expression of the peptidesaccording to the present invention are the murine EL4 and P8.15 cells.For expression of said peptides human BLM cells (Katano et al., 1984, J.Cancer Res. Clin. Oncol. 108, 197) are especially suited because theyalready are able to express the MHC molecule HLA-A2.1.

The peptides according to the present invention can be used in a vaccinefor the treatment of melanoma.

In addition to an immunogenically effective amount of the activepeptide, the vaccine may contain a pharmaceutically acceptable carrieror diluent.

The immunogenicity of the peptides of the invention, especially theoligopeptides, can be enhanced by cross-linking or by coupling to animmunogenic carrier molecule (i.e., a macromolecule having the propertyof independently eliciting an immunological response in a patient, towhich the peptides of the invention can be covalently linked) or if partof a protein.

Covalent coupling to the carrier molecule can be carried out usingmethods well known in the art, the exact choice of which will bedictated by the nature of the carrier molecule used. When theimmunogenic carrier molecule is a protein, the peptides of the inventioncan be coupled, e.g., using water soluble carbodiimides such asdicyclohexylcarbodiimide, or glutaraldehyde.

Coupling agents such as these can also be used to cross-link thepeptides to themselves without the use of a separate carrier molecule.Such cross-linking into polypeptides or peptide aggregates can alsoincrease immunogenicity.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include stabilizers such as SPGA, carbohydrates(e.g., mannose, sorbitol, mannitol, starch, sucrose, glucose, dextran),proteins such as albumin or casein, protein containing agents such asbovine serum or skimmed milk and buffers (e.g., phosphate buffer).

Optionally, one or more compounds having adjuvant activity may be addedto the vaccine. Suitable adjuvants are for example aluminium hydroxide,phosphate or oxide, oil-emulsions (e.g. of Bayol F® or Marcol 52®),saponins or vitamin-E solubilisate.

Dendritic cells are professional APC that express mannose receptor usedto take up antigen thus facilitating antigen processing.

The vaccine according to the present invention can be given inter aliaintravenously, intraperitoneally, intranasally, intradermally,subcutaneously or intramuscularly.

The useful effective amount to be administered will vary depending onthe age and weight of the patient and mode of administration of thevaccine.

The vaccine can be employed to specifically obtain a T cell response,but it is also possible that a B cell response is elicited aftervaccination. If so, the B cell response leads to the formation ofantibodies against the peptide of the vaccine, which antibodies will bedirected to the source of the antigen production, i.e., the tumor cells.This is an advantageous feature, because in this way the tumor cells arecombated by responses of both the immunological systems.

Both immunological systems will even be more effectively triggered whenthe vaccine comprises the peptides as presented in an MHC molecule by anantigen presenting cell (APC). Antigen presentation can be achieved byusing monocytes, macrophages, interdigitating cells, Langerhans cellsand especially dendritic cells, loaded with one of the peptides of theinvention or loading with protein including peptide or manosylatedprotein. Loading of the APCs can be accomplished by bringing thepeptides of the invention into or in the neighborhood of the APC, but itis more preferable to let the APC process the complete gp100 antigen. Inthis way a presentation is achieved which mimics the in vivo situationmost realistically. Furthermore, the MHC used by the cell is of the typewhich is suited to present the epitope.

An overall advantage of using APCs for the presentation of the epitopesis the choice of APC cell that is used in this respect. It is known fromdifferent types of APCs that there are stimulating APCs and inhibitingAPCs.

Preferred APCs include, but are not limited to, the listed cell types,which are so-called “professional” antigen presenting cells,characterized in that they have co-stimulating molecules, which have animportant function in the process of antigen presentation. Suchco-stimulating molecules are, for example, B7, CD25, CD40, CD70, CTLA-4or heat stable antigen (Schwartz, 1992, Cell 71, 1065-1068).

Fibroblasts, which have also been shown to be able to act as an antigenpresenting cell, lack these co-stimulating molecules.

It is also possible to use cells already transfected with a cloningvehicle harboring the information for the melanocyte peptide analoguesand which are cotransfected with a cloning vehicle which comprises thenucleotide sequence for an MHC class I molecule, for instance thesequence coding for HLA A2.1, HLA A1 or HLA A3.1. These cells will actas an antigen presenting cell and will present peptide analogues in theMHC class I molecules which are expressed on their surface. It isenvisaged that this presentation will be enhanced, when the cell is alsocapable of expressing one of the above-mentioned co-stimulatingmolecules (in particular B7 (B7.1, B7.2), CD40), or a molecule with asimilar function (e.g., cytokines transfected in cell line). Thisexpression can be the result of transformation or transfection of thecell with a third cloning vehicle having the sequence information codingfor such a co-stimulating molecule, but it can also be that the cellalready was capable of production of co-stimulating molecules.

Instead of a vaccine with these cells, which next to the desiredexpression products, also harbor many elements which are also expressedand which can negatively affect the desired immunogenic reaction of thecell, it is also possible that a vaccine is composed with liposomeswhich expose MHC molecules loaded with peptides, and which, forinstance, are filled with lymphokines. Such liposomes will trigger animmunologic T cell reaction.

By presenting the peptide in the same way as it is also presented invivo, an enhanced T cell response will be evoked. Furthermore, by thenatural adjuvant working of the relatively large, antigen presentingcells also a B cell response is triggered. This B cell response willalso lead to the formation of antibodies directed to the peptide-MHCcomplex. This complex is especially found in tumor cells, where it hasbeen shown that in the patient epitopes of gp100 are presentednaturally, which are thus able to elicit a T cell response. It is thisnaturally occurring phenomenon which is enlarged by the vaccination ofAPCs already presenting the peptides of the invention. By enlarging notonly an enlarged T cell response will be evoked, but also a B cellresponse which leads to antibodies directed to the MHC-peptide complexwill be initiated.

The vaccines according to the invention can be enriched by numerouscompounds which have an enhancing effect on the initiation and themaintenance of both the T cell and the B cell response aftervaccination.

In this way, addition of cytokines to the vaccine will enhance the Tcell response. Suitable cytokines are for instance interleukins, such asIL-2, IL-4, IL-7, or IL-12, GM-CSF, RANTES, MIP-α, and tumor necrosisfactor, and interferons, such as IFN- or the chemokins.

In a similar way, antibodies against T cell surface antigens, such asCD2, CD3, CD27 and CD28 will enhance the immunogenic reaction.

Also, the addition of helper epitopes to stimulate CD4⁺ helper cells orCD8⁺ killer cells augments the immunogenic reaction. Alternatively, alsohelper epitopes from other antigens can be used, for instance from heatshock derived proteins or cholera toxin.

Another part of the invention is formed by using reactive tumorinfiltrating lymphocytes (TILs) directed against the peptides accordingto the present invention. In this method, the first step is taking asample from a patient. This is usually done by resection of a tumordeposit under local anesthesia. The TILs present in this specimen arethen expanded in culture for four to eight weeks, according to knownmethods (Topalian et al., 1987, J. Immunol. Meth. 102, 127-141). Duringthis culture, the TILs are then checked for reactivity with the peptidesaccording to the present invention or gp100-protein. The TILs whichrecognize the antigen are isolated and cultured further.

The reactive tumor infiltrating lymphocytes which are obtained throughthis method, also form part of the invention. An example of such TILcell line, designated TIL 1200, has been found which specifically reactswith gp100 and its epitopes. This TIL 1200 cell line also expresses theMHC molecule HLA-A2.1. Furthermore, expression of TCR α/β, CD3 and CD8by this cell line has been demonstrated. Furthermore, TIL 1200recognizes transfectants expressing both HLA-A2.1 and gp100.

TIL 1200 and other TILs recognizing gp100 are suited for treatment ofmelanoma patients. For such treatment, TILs may be cultured as statedabove, and they are given back to the patients by an intravenousinfusion. The success of treatment can be enhanced by pre-treatment ofthe tumor bearing host with either total body radiation or treatmentwith cyclophosphamide and by the simultaneous administration ofinterleukin-2 (Rosenberg et al., 1986, Science 223, 1318-1321).

The TILs infused back to the patient are preferably autologous TILs(i.e., derived from the patient's own tumor) but also infusion withallogenic TILs can be imagined.

A further use of the TILs obtained by the method as described above isfor in vivo diagnosis. Labeling of the TILs, for instance with ¹¹¹In(Fisher et al., 1989, J. Clin. Oncol. 7, 250-261) or any other suitablediagnostic marker, renders them suited for identification of tumordeposits in melanoma patients.

Another part of the invention is formed by the T cell receptor (TCR)expressed by reactive CTLs directed against the peptides according tothis invention or the gp100-protein. As is well known in the art, theTCR determines the specificity of a CTL. Therefore, the cDNA encodingthe TCR, especially its variable region, can be isolated and introducedinto T cells, thereby transferring anti-tumor activity to any T cell.Especially introduction of such a TCR into autologous T cells andsubsequent expansion of these T cells will result in large numbers ofCTL suitable for adoptive transfer into the autologous patient.

Cells harboring this T cell receptor can also be used for vaccinationpurposes.

A vaccine can also be composed from melanoma cells capable of expressionof the peptides according to the present invention. It is possible toisolate these cells from a patient, using specific antibodies, such asNKI-beteb (directed against gp100), but is also possible to produce suchmelanoma cells from cultured melanoma cell lines, which either arenatural gp100-producers or have been manipulated genetically to producethe peptides according to the present invention. These cells can beirradiated to be non-tumorogenic and infused (back) into the patient. Toenhance the immunologic effect of these melanoma cells it is preferredto alter them genetically to produce a lymphokine, preferablyinterleukin-2 (IL-2) or granulocyte-macrophage colony stimulation factor(GM-CSF). Peptide⁺/gp100⁺ melanoma cells can be transfected with acloning vehicle having the sequence coding for the production of IL-2 orGM-CSF.

Infusion of such a vaccine into a patient will stimulate the formationof CTLs.

Another type of vaccination having a similar effect is vaccination withpure DNA, for instance the DNA of a vector or a vector virus having theDNA sequence encoding the peptides according the present invention (bothhomologues and heterologues (chimeric protein) or repetitive). Onceinjected, the virus will infect or the DNA will be transformed to cellswhich express the antigen or the peptide(s).

Antibodies directed against the peptides according to the presentinvention are also part of the invention.

Monospecific antibodies to these peptides can be obtained by affinitypurification from polyspecific antisera by a modification of the methodof Hall et al. (1984, Nature 311, 379-387). Polyspecific antisera can beobtained by immunizing rabbits according to standard immunizationschemes.

Monospecific antibody as used herein is defined as a single antibodyspecies or multiple antibody species with homogeneous bindingcharacteristics for the relevant antigen. Homogeneous binding as usedherein refers to the ability of the antibody species to bind to ligandbinding domain of the invention.

The antibody is preferably a monoclonal antibody, more preferably ahumanized monoclonal antibody.

Monoclonal antibodies can be prepared by immunizing inbred mice,preferably Balb/c with the appropriate protein by techniques known inthe art (Köhler, G. and Milstein C., 1975, Nature 256, 495-497).Hybridoma cells are subsequently selected by growth in hypoxanthine,thymidine and aminopterin in an appropriate cell culture medium such asDulbecco's modified Eagle's medium (DMEM). Antibody producing hybridomasare cloned, preferably using the soft agar technique of MacPherson(1973, Tissue Culture Methods and Applications, Kruse and Paterson,eds., Academic Press). Discrete colonies are transferred into individualwells of culture plates for cultivation in an appropriate culturemedium. Antibody producing cells are identified by screening with theappropriate immunogen. Immunogen positive hybridoma cells are maintainedby techniques known in the art. Specific anti-monoclonal antibodies areproduced by cultivating the hybridomas in vitro or preparing ascitesfluid in mice following hybridoma injection by procedures known in theart.

It may be preferred to use humanized antibodies. Methods for humanizingantibodies, such as CDR-grafting, are known (Jones et al., 1986, Nature321, 522-525). Another possibility to avoid antigenic response toantibodies reactive with polypeptides according to the invention is theuse of human antibodies or fragments or derivatives thereof.

Human antibodies can be produced by in vitro stimulation of isolatedB-lymphocytes, or they can be isolated from (immortalized) B-lymphocyteswhich have been harvested from a human being immunized with at least oneligand binding domain according to the invention.

Antibodies as described above can be used for the passive vaccination ofmelanoma patients. A preferred type of antibodies for this kind ofvaccine are antibodies directed against the above-mentioned peptidespresented in connection with the MHC molecule. To produce these kind ofantibodies immunization of peptides presented by APCs is required. Suchan immunization can be performed as described above. Alternatively,human antibodies to peptide-MHC complexes can be isolated from patientstreated with a vaccine consisting of APCs loaded with one of saidpeptides.

The antibodies, which are formed after treatment with one of thevaccines of the invention can also be used for the monitoring of saidvaccination. For such a method, serum of the patients is obtained andthe antibodies directed to the peptide with which has been vaccinatedare detected. Knowing the antibody titre from this detection, it can bejudged if there is need for a boost vaccination.

Specific detection of said antibodies in the serum can be achieved bylabeled peptides. The label can be any diagnostic marker known in thefield of in vitro diagnosis, but most preferred (and widely used) areenzymes, dyes, metals and radionuclides, such as ⁶⁷Ga, ^(99m)Tc, ¹¹¹In,^(113m)In, ¹²³I, ¹²⁵I, or ¹³¹I.

The radiodiagnostic markers can be coupled directly to the peptides ofthe invention or through chelating moieties which have been coupled tothe peptide directly or through linker or spacer molecules. Thetechnique of coupling of radionuclides to peptides or peptide-likestructures is already known in the field of (tumor) diagnostics from thenumerous applications of labeled antibodies used both in in vivo and inin vitro tests.

Direct labeling of peptides can, for instance, be performed as describedin the one-vial method (Haisma et al., 1986, J. Nucl. Med. 27, 1890). Ageneral method for labeling of peptides through chelators, with orwithout linker or spacer molecules, has, for instance, been described inU.S. Pat. Nos. 4,472,509 and 4,485,086. Chelators using a bicyclicanhydride of DTPA have been disclosed in Hnatowich et al. (1983, J.Immunol. Meth. 65, 147-157). Coupling through diamide dimercaptidecompounds has been disclosed in EP 188,256.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The present invention is further described by way of examples withreference to the accompanying figures, in which:

FIG. 1. Target cell sensitization of alanine replacement epitopes. (A)Chromium labeled T2 target cells were preincubated for 1 hour withvarious amounts of the indicated alanine-substituted epitope-analogues.Melan-A/MART-1 27-35-reactive TIL 1235 lymphocytes were added at aneffector to target ratio of 20. (B) Target cell sensitization ofalanine-substituted gp100 154-162-analogues was analyzed usinggp100-reactive TIL 1200 lymphocytes at an effector to target ratio of20.

FIG. 2. Target cell sensitization of N-terminal anchor-replacementepitopes. Chromium release experiments were performed as in FIG. 1. (A)Melan-A/MART-1 27-35-reactive TIL 1235 lymphocytes were used to assaytarget cell sensitization by the Melan-A/MART-1 27-35 analogues. (B)Gp100 154-162-reactive TIL 1200 lymphocytes were used to assay targetcell sensitization by the gp100 154-162-analogues.

FIG. 3. Immunogenicity of gp100 154-162 epitope-analogues inHLA-A*0201/K^(b) transgenic mice. Bulk CTL obtained from immunized micewere tested for lytic activity using chromium labeled Jurkat A2/K^(b)target cells that were preincubated with no peptide, 10 mM wild typegp100 154-162 or 10 mM of the epitope-analogue used to immunize themice. For each peptide the mean specific lysis of bulk CTL of theresponding mice is shown. Standard deviations never exceeded 15% of themean value. One representative experiment out of two is shown.

FIG. 4. Peptide specific reactivity of in vitro induced epitope-analoguespecific CTL cultures. Chromium-labeled HLA-A*0201⁺ T2 target cells werepre-incubated with 10 mM of an irrelevant HLA-A*0201-binding peptide, 10mM wild type gp100 154-162 or 10 mM of the epitope-analogue used for CTLinduction. The different CTL cultures were added at an effector totarget ratio of 20:1. One representative experiment out of two is shown.

FIG. 5. Epitope-analogue induced CTL cultures specifically lyse melanomacells endogenously presenting the wild type epitope. Chromium-labeledHLA-A2.1⁺ BLM and Mel 624 melanoma cells were used as target cells. BLMcells lack expression of gp100. The different CTL cultures were added atan effector to target ratio of 20:1. One representative experiment outof two is shown.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods CellCulture.

The HLA-A*0201⁺ melanoma line BLM was cultured as described previously(Bakker et al, 1994, J. Exp. Med. 179:1005). TIL 1200 and TIL 1235lymphocytes were cultured as was reported previously (Kawakami et al.,1992, J. Immunol. 148:638). T2 cells (Salter et al., 1985,Immunogenetics. 21:235) and HLA-A*0201⁺ B lymphoblastoid JY cells weremaintained in Iscoves medium (Gibco, Paisley, Scotland UK) supplementedwith 5% FCS (BioWhittaker, Verviers, Belgium). Jurkat A*0201/K^(b) cells(Irwin et al., 1989, J. Exp. Med. 170:1091) expressing theHLA-A*0201/K^(b) chimeric molecule were cultured in Iscoves medium with5% FCS supplemented with 0.8 mg/ml G418 (Gibco, Paisley, Scotland UK).

HLA-A*0201⁺ Lymphocytes.

Healthy caucasian volunteers were phenotyped HLA-A2 by flow cytometryusing mAbs BB7.2 (Parham et al., 1981, Hum. Immunol. 3:277) and MA2.1(Parham et al., 1978, Nature 276:397). The donors underwentleukapheresis and PBMC were isolated by Ficoll/Hypaque density gradientcentrifugation. The cells were cryopreserved in aliquots of 4×10⁷ PBMC.

Transgenic Mice

HLA-A*0201/K^(b) transgenic mice were used (animal distributor HarlanSprague Dawley, Inc., Indianapolis, USA). Mice were held under cleanconventional conditions. The transgenic mice express the product of theHLA-A*0201/K^(b) chimeric gene in which the α3 domain of the heavy chainis replaced by the corresponding murine H-2 K^(b) domain while leavingthe HLA-A*0201 at and a2 domains unaffected (Vitiello et al., 1991, J.Exp. Med. 1007). This allows the murine CD8 molecule on the murine CD8⁺T lymphocytes to interact with the syngeneic α3 domain of the hybrid MHCclass I molecule.

Peptides.

For induction of CTL and chromium-release assays, peptides weresynthesized with a free carboxy-terminus by Fmoc peptide chemistry usingan ABIMED multiple synthesizer. All peptides were >90% pure as indicatedby analytical HPLC. Peptides were dissolved in DMSO and stored at −20°C.

HLA-A*0201 Upregulation on T2 Cells.

Peptide-induced HLA-A*0201 upregulation on T2 cells was performed asdescribed previously (Nijman et al., 1993, Eur. J. Immunol. 23:1215).Briefly, peptides were diluted from DMSO stocks to variousconcentrations (final DMSO concentration 0.5%) and were incubatedtogether with 10⁵ T2 cells for 14 hours at 37° C., 5% C0₂ in serum-freeIscoves medium in a volume of 100 ml in the presence of 3 mg/ml humanβ2-microglobulin (Sigma, St Louis, Mo.). Stabilization of HLA-A*0201molecules at the cell surface of T2 cells was analyzed by flow cytometryusing anti-HLA-A2 mAb BB7.2 (Parham et al., 1981, Hum. Immunol. 3:277).The Fluorescence Index is expressed as: (experimental mean fluorescence÷background mean fluorescence)−1. The background mean fluorescence valueswere obtained by incubating T2 cells with a HLA-A*0201 non-bindingpeptide at similar concentrations.

Competition Based HLA-A*0201 Peptide-Binding Assay.

Peptide-binding to HLA-A*0201 was analyzed using HLA-A*0201⁺ JY cells aswas described previously (van der Burg et al., 1995, Hum. Immunol.44:189). Briefly, mild-acid treated JY cells were incubated with 150 nMFluorescein (FL)-labeled reference peptide (FLPSDC(-FL)FPSV) and withseveral concentrations of competitor peptide for 24 hours at 4° in thepresence of 1.0 mg/ml β2-microglobulin (Sigma, St. Louis, Mo.).Subsequently, the cells were washed, fixed with paraformaldehyde andanalyzed by flow cytometry. The mean-fluorescence (MF) obtained in theabsence of competitor peptide was regarded as maximal binding andequated to 0%; the MF obtained without reference peptide was equated to100% inhibition. % inhibition of binding was calculated using theformula: (1-(MF 150 nM reference & competitor peptide−MF no referencepeptide)÷(MF 150 nM reference peptide-MF no reference peptide))×100%.The binding capacity of competitor peptides is expressed as theconcentration needed to inhibit 50% of binding of the FL-labeledreference peptide (IC₅₀.

Measurement of MHC-Peptide Complex Stability at 37° C.

Measurement of MHC-peptide complex stability was performed. HLA-A*0201⁺homozygous JY cells were treated with 10⁴ M emetine (Sigma, St. Louis,USA) for 1 hour at 37° C. to stop de novo synthesis of MHC class Imolecules. The cells were then mild-acid treated and subsequently loadedwith 200 mM of peptide for 1 hour at room temperature. Thereafter, thecells were washed twice to remove free peptide and were incubated at 37°C. for 0, 2, 4 and 6 hours. Subsequently, the cells were stained usingmAb BB7.2 (Parham et al., 1981, Hum. Immunol. 3:277), fixed withparaformaldehyde and analyzed by flow cytometry.

CTL Induction in HLA-A*0201/K^(b) Transgenic Mice. Groups of 3HLA-A*0201/K^(b) transgenic mice were injected subcutaneously in thebase of the tail vein with 100 mg peptide emulsified in WA in thepresence of 140 mg of the H-2 I-A^(b)-restricted HBV coreantigen-derived T helper epitope (128-140; sequence TPPAYRPPNAPIL)(Milich et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:1610). After 11days, mice were sacrificed and spleen cells (30×10⁶ cells in 10 ml) wererestimulated in vitro with peptide-loaded syngeneic irradiatedLPS-stimulated B cell lymphoblasts (ratio 4:1). At day 6 of culture, thebulk responder populations were tested for specific lytic activity.

HLA-A*0201⁺ Donor Derived CTL Induction In Vitro

Using thawed PBMC, dendritic cells were generated according theprocedure of Romani et al. (Romani et al., 1994, J. Exp. Med. 180:83) aswas described previously (Bakker et al., 1995, Cancer Res. 55:5330).Before the onset of culture, dendritic cells were loaded with 50 mM ofpeptide. Autologous CM⁺ enriched responder T lymphocytes were preparedby adhering thawed PBMC for 2 hours and by subsequent partial depletionof the non-adherent fraction of CD4⁺ T cells using the anti-CD4 mAbRIV-7 (Leerling et al., 1990, Dev. Biol. Stand. 71:191) andSheep-anti-Mouse-IgG coated magnetic beads (Dynal, Oslo, Sweden). At theonset of stimulation, 2×10⁵ peptide-loaded DC and 2×10⁶ responder cellswere co-cultured per well of a 24-well tissue culture plate (Costar,Badhoevedorp, The Netherlands) in 2 ml of Iscoves medium containing 5%pooled human AB⁺ serum, 10³ U/ml IL-6 (Sandoz, Basel, Switzerland) and 5ng/ml IL-12.

On day 8 and day 15, the responder populations were restimulated usingpeptide-pulsed dendritic cells as stimulator cells. The cultures werepropagated in medium containing IL-2 (Cetus Corp., Emeryville, Calif.)and IL-7 (Genzyme, Cambridge, Mass.) at final concentrations of 10 U/mland 5 ng/ml respectively. Weekly hereafter the cultures wererestimulated using adherent peptide-pulsed PBMC as was describedpreviously (Bakker et al., 1995 , Cancer Res. 55:5330). Responderpopulations were tested for specific lytic activity after at least 4rounds of restimulation.

Chromium-Release Assay.

Chromium release assays were performed as described previously (Bakkeret al., 1994, J. Exp. Med. 179:1005). Briefly, 10⁶ target cells wereincubated with 100 mCi Na₂ ⁵¹CrO₄ (Amersham, Bucks, UK) for 1 hour.Various amounts of effector cells were then added to the target cells intriplicate wells of U bottomed microliter plates (Costar, Badhoevedorp,The Netherlands) in a final volume of 150 ml. In peptide recognitionassays, target cells were pre-incubated with various concentrations ofpeptide for 30 or 60 min at 37° C. in a volume of 100 ml prior to theaddition of effector cells. After 5 h of incubation, part of thesupernatant was harvested and its radioactive content was measured. Themean percentage specific lysis of triplicate wells was calculated usingthe formula: % specific lysis=((experimental release−spontaneousrelease)÷(maximal release−spontaneous release))×100.

Example 1 Identification of Amino Acid Residues Engaged in HLA-A*0201Binding and/or TCR Interactions for the Melan-A/MART-1 27-35 and thegp100 154-162 Epitopes

The Melan-A/MART-1 27-35 and the gp100 154-162 epitopes have beenidentified using HLA-A*0201 restricted TIL lines derived from metastaticmelanomas. The Melan-A/MART-1 27-35 epitope was found to be the nominalepitope capable of triggering the Melan-A/MART-1 specific TIL 1235 linewhen presented on HLA-A*0201⁺ target cells (Kawakami et al., 1994. J.Exp. Med. 180:347). Among a panel of peptides ranging from 8-mers to11-mers located around gp100 amino acids 155-161, we identified the9-mer 154-162 as the peptide most efficient in sensitizing HLA-A*0201⁺target cells for lysis by the gp100 reactive TIL 1200 line (Bakker etal., 1995, Int. J. Cancer 62:97). Both the Melan-A/MART-1 27-35 9-merand the gp100 154-162 9-mer have now been eluted from the cell surfaceof HLA-A*0201⁺ melanoma cells, and were identified by tandemmass-spectroscopy, indicating that they are indeed the nominal epitopesendogenously presented in HLA-A*0201. To identify amino acid residues inboth epitopes engaged in HLA-A*0201 binding and/or TCR interactions,epitope-analogues were synthesized in which the native amino acid wasreplaced by an alanine residue. In case alanine residues were present inthe wild type epitope, they were substituted for the amino acid glycine.The substituted peptides were assayed for binding to HLA-A*0201 by meansof an indirect binding assay using the processing defective cell line T2(Nijman et al., 1993, Eur. J. Immunol. 23:1215). All substitutions inthe Melan-A/MART-1 epitope resulted in a nearly complete loss in thecapability to stabilize HLA-A*0201 molecules at the cell surface of T2cells (Table I). When the Melan-A/MART-1 27-35 analogues were used atmicromolar concentrations to sensitize HLA-A*0201⁺ target cells forlysis by Melan-A/MART-1-specific CTL, we observed a decrease in targetcell lysis for the alanine replacements at positions 4 to 7 of theepitope (Table I). In addition, the glycine substitution at position 2resulted in decreased CTL reactivity. The amino acids at these positionsin the Melan-A/MART-1 27-35 epitope are therefore most likely involvedin TCR interactions.

In case of the gp100 154-162 epitope decreased HLA-A*0201 affinity ofepitope-analogues was only observed for the alanine substitutions atposition 3 and 9 (Table 1). With respect to T cell recognition, alaninesubstitutions at positions 5, 6 and 7 of the epitope were not allowed,indicating that amino acids at these positions are critical contactresidues within this epitope for the TCR.

Subsequently, the epitope-analogues that induced reactivity atmicromolar concentrations were titrated to evaluate their relativeability to sensitize T2 target cells for lysis by the relevant CTL (FIG.1). In all cases the epitope-analogues were similar or inferior comparedto the wild type epitope in their sensitizing capacity, except for thealanine substitution at position 8 of the gp100 154-162 epitope.Surprisingly, this peptide was able to induce target cell lysis bygp100-reactive CTL even at a tenfold lower concentration.

Example 2 N-Terminal Anchor Residue Replacements in Both the gp100154-162 and the Melan-A/MART-1 27-35 Epitopes Result in ImprovedAffinity for HLA-A*0201

Since both the Melan-A/MART-1 27-35 and the gp100 154-162 epitopes havenon-conventional N-terminal anchoring residues, we replaced theseresidues for the common HLA-A*0201 anchoring residues V, L, I or M(Drijthout et al., 1995, Hum. Immunol. 43:1). Subsequently, we testedthese peptides for HLA-A*0201 binding and their ability to sensitizetarget cells for lysis by the relevant CTL. Apart from the methioninesubstitution, all anchor residue replacements in the Melan-A/MART-1epitope resulted in significantly improved binding to HLA-A*0201 (TableII). HLA-A*0201⁺ target cells loaded with these peptides at aconcentration of 1 mM were recognized by the Melan-A/MART-1 reactiveCTL, except for the methionine substituted epitope (Table II). Althoughthis peptide did bind to HLA-A*0201 at a level comparable to the wildtype epitope, it failed to induce CTL reactivity. Titration experimentsusing the Melan-A/MART-1 anchor replacement peptides demonstrated thatthese epitope-analogues were inferior to wild type in sensitizing targetcells for lysis by TIL 1235 (FIG. 2).

Using the T2 assay all gp100 154-162 anchor replacement peptides exceptthe methionine substituted epitope showed HLA-A*0201 binding comparableto the wild type epitope (Table II). Interestingly, these peptides wererecognized by TIL 1200 when loaded on target cells at tenfold lowerconcentrations compared to the wild type peptide (FIG. 2), while themethionine substituted peptide showed no difference. These findingsdemonstrate that amino acid substitutions within the native epitope canresult in improved T cell recognition.

Example 3 Improved Target Cell Sensitization by gp100 154-162 EpitopeAnalogues Correlates with Increased Affinity for HLA-A*0201

To assess whether the augmented CTL recognition of the substituted gp100154-162 epitopes could be attributed to improved HLA-A*0201 affinity,the HLA-A*0201 binding capacity of these peptides was tested now using amore sensitive cell-bound HLA-A*0201 binding assay based on competitionof a labeled reference peptide with the peptides of interest (van derBurg et al., 1995, Hum. Immunol. 44:189). HLA-A*0201 binding-affinitiesobtained with this assay demonstrated that all peptides that were ableto sensitize target cells for lysis by TIL 1200 at tenfold lowerconcentrations compared to wild type, also bound with higher affinity toHLA-A*0201 (Table III). In addition to the N-terminal anchorsubstitutions, replacement of a polar residue for a hydrophobic residueadjacent to the C-terminal anchoring position also resulted in anepitope-analogue with improved HLA-A*0201 affinity (KTWGQYWAV (SEQ IDNO: 1)), apparently without affecting TCR recognition. Measurement ofMHC class I-peptide complex dissociation rates demonstrated that theepitope-analogues tested are at least equally stable when compared towild type (Table III). All peptides tested showed a DT₅₀ (the timerequired for 50% of the complexes to decay) longer than 4 hours.Peptides with DT₅₀ values of ≧3 hours were immunogenic inHLA-A*0201/K^(b) transgenic mice. Taken together, these data indicatethat the gp100 154-162 epitope-analogues may have similar or increasedimmunogenicity compared to wild type gp100 154-162.

Example 4 Immunogenicity of gp100 154-162 Epitope-Analogues inHLA-A*0201/K^(b) Transgenic Mice

In order to determine the in vivo immunogenicity of the gp100 154-162epitope-analogues of which the MHC class I binding-affinity anddissociation rate was measured. HLA-A*0201/K^(b) transgenic mice werevaccinated with the gp100 154-162 wild type epitope, with theepitope-analogues KTWGQYWAV (SEQ ID NO: 1), KVWGQYWQV (SEQ ID NO: 2),KLWGQYWQV (SEQ ID NO: 3) or KIWGQYWQV (SEQ ID NO: 4), or with a controlpeptide (HBV core 18-27: FLPSDDFPSV (SEQ ID NO: 6)). The generation ofthese transgenic mice (Vitiello et al., 1991. J. Exp. Med. 173:1007) andtheir use to analyze in vivo immunogenicity have been describedpreviously (Ressing et al., 1995, J. Immunol. 154:5934; Sette et al.,1994, J. Immunol. 153:5586). As shown in FIG. 3, the gp100 154-162epitope-analogues KTWGQYWAV (SEQ ID NO: 1), KVWGQYWQV (SEQ ID NO: 2),and KLWGQYWQV (SEQ ID NO: 3), very efficiently induced a CTL response.To a lesser extent also the epitope-analogue KIWGQYWQV (SEQ ID NO: 4)and the wild type gp100 154-162 were able to elicit a CTL response. BulkCTL derived from mice vaccinated with the gp100 154-162epitope-analogues specifically lysed Jurkat A*0201/K^(b) cells loadedwith both the peptide used for vaccination and the wild type epitope.Interestingly, CTL bulk cultures raised against the epitope-analoguesall recognized target cells pulsed with the wild type epitope equallywell or better compared to target cells pulsed with epitope-analoguesused for vaccination. Thus, all gp100 154-162 epitope-analogues testedwere immunogenic in HLA-A*0201/K^(b) transgenic mice, and elicited CTLdisplaying cross-reactivity with the native gp100 154-162 epitope.

Example 5 In Vitro Induction of gp100 154-162 Epitope-Analogue SpecificHuman CTL Displaying Cross-Reactivity with Endogenously HLA-A*9201Presented Wild Type gp100 154-162

Next, we performed in vitro CTL induction assays to assess whetherwithin the T cell repertoire of HLA-A*0201⁺ healthy donors precursor Tlymphocytes were present capable of recognizing gp100 154-162epitope-analogues. In order to achieve this, we initiated cultures ofpeptide-loaded dendritic cells together with autologous responder Tlymphocytes as described previously (Bakker et al., 1995, Cancer Res.55:5330). After several rounds of restimulation, responder T cells weretested for cytotoxic activity (FIG. 4). All bulk CTL populations raisedagainst the gp100 154-162 epitope-analogues, KTWGQYWAV (SEQ ID NO: 1),KVWGQYWQV (SEQ ID NO: 2), KLWGQYWQV (SEQ ID NO: 3) and KIWGQYWQV (SEQ IDNO: 4), efficiently lysed HLA-A*0201⁺ T2 target cells incubated with thepeptides used for CTL induction. Only low background lysis was observedin the presence of an irrelevant peptide. In addition, these gp100154-162 epitope-analogue reactive CTL efficiently lysed T2 target cellsincubated with wild type gp100 154-162. To address the question whetherthese CTL responder populations could also recognize endogenouslyprocessed and presented wild type epitope, we performed chromium-releaseexperiments using HLA-A*0201⁺ melanoma cell lines BLM and MeI 624 astargets. BLM cells have lost expression of the gp100 antigen, both atthe protein and at the mRNA level (Adema et al., 1993, Am. J. Pathol.143:1579). As shown in FIG. 5, all peptide-induced CTL cultures lysedthe antigen expressing MeI 624 cells, whereas no or background lysis wasobserved against antigen negative BLM cells. TNF release by theanti-gp100 154-162 analogue CTL further demonstrated the reactivity ofthese CTL with endogenously presented wild type gp100 154-162 (data notshown). These data show that the four different CTL cultures inducedusing gp100 154-162 epitope-analogue loaded dendritic cells, allrecognized the native gp100 154-162 epitope endogenously processed andpresented by HLA-A*0201⁺ Mel 624 cells.

TABLE IHLA-A*0201-binding and target cell sensitization of alanine-replacement epitopes.target cell target HLA-A*0201 lysis by HLA-A*0201 cell stabilization^(a)TIL stabilization lysis by Melan A/MART-1 27-35 50 μM 25 μM 1235^(b)gp100 154-162 50 μM 25 μM TIL 1200 YLEPGPVTA^(c) (SEQ ID NO: 7) 2.262.12 −3 YLEPGPVTA (SEQ ID NO: 7) 3 AAGIGILTV (SEQ ID NO: 8) 1.20 1.11 40KTWGQYWQV (SEQ ID NO: 6) 2.06 1.40 67 GAGIGILTV (SEQ ID NO: 10) 1.071.11 52 ATWGQYWQV (SEQ ID NO: 11) 1.94 1.42 75 AGGIGILTV (SEQ ID NO: 12)0.96 1.05 6 KAWGQYWQV (SEQ ID NO: 13) 1.57 1.20 64AAAIGILTV (SEQ ID NO: 14) 0.98 0.99 13 KTAGQYWQV (SEQ ID NO: 15) 1.171.02 58 AAGAGILTV (SEQ ID NO: 16) 0.93 0.97 0 KTWAQYWQV (SEQ ID NO: 17)1.45 1.13 63 AAGIAILTV (SEQ ID NO: 18) 1.01 1.01 4KTWGAYWQV (SEQ ID NO: 19) 1.59 1.25 9 AAGIGALTV (SEQ ID NO: 20) 0.931.00 2 KTWGQAWQV (SEQ ID NO: 21) 1.42 1.15 7 AAGIGIATV (SEQ ID NO: 22)1.10 1.13 6 KTWGQYAQV (SEQ ID NO: 23) 1.31 1.14 −2AAGIGILAV (SEQ ID NO: 24) 1.05 1.01 11 KTWGQYWAV (SEQ ID NO: 1) 1.721.35 73 AAGIGILTA (SEQ ID NO: 25) 1.00 1.03 26 KTWGQYWQA (SEQ ID NO: 26)1.08 1.02 76 ^(a)Binding of peptides to HLA-A2.1 was analyzed using theprocessing-defective T2 cell line at the indicated peptideconcentrations. Numbers indicate Fluorescence Index: experimental meanfluorescence divided by the mean fluorescence that is obtained when T2cells are incubated with an HLA-A2.1 non-binding peptide at a similarconcentration. ^(b)Numbers indicate % specific lysis by the relevant TILlines at an E:T ratio of 20:1. Chromium-labeled T2 target cells werepreincubated for 90 min with 1 μM of peptide. Chromium release wasmeasured after 5 hours of incubation. ^(c)gp100 280-288.

TABLE IIHLA-A*0201-binding and target cell sensitization of N-terminal anchor-replacement epitopes.HLA-A*0201 HLA-A*0201 stabilization^(a) target cell stabilizationtarget cell 50 25 lysis by  50 25 lysis by Melan A/MART-1 27-35 μM μMTIL 1235^(b) gp100 154-162 μM μM TIL 1200 YLEPGPVTA^(c) (SEQ ID NO: 7)2.26 2.12 −1 YLEPGPVTA (SEQ ID NO: 7) 3 AAGIGILTV (SEQ ID NO: 8) 1.201.11 40 KTWGQYWQV (SEQ ID NO: 9) 2.06 1.40 67 AVGIGILTV (SEQ ID NO: 27)1.62 1.36 27 KVWGQYWQV (SEQ ID NO: 2) 2.13 1.57 69ALGIGILTV (SEQ ID NO: 28) 2.21 1.93 16 KLWGQYWQV (SEQ ID NO: 3) 2.191.55 65 AMGIGILTV (SEQ ID NO: 29) 1.18 1.05 6 KMWGQYWQV (SEQ ID NO: 35)1.73 1.28 57 AIGIGLTV (SEQ ID NO: 30) 1.58 1.29 27KIWGQYWQV (SEQ ID NO: 4) 2.00 1.43 68 ^(a)Binding of peptides toHLA-A2.1 was analyzed using the processing-defective T2 cell line at theindicated peptide concentrations. Numbers indicate Fluorescence Index:experimental mean fluorescence divided by the mean fluorescence that isobtained when T2 cells are incubated with an HLA-A2.1 non-bindingpeptide at a similar concentration. ^(b)Numbers indicate % specificlysis by the relevant TIL lines at an E:T ratio of 20:1.Chromium-labeled T2 target cells were preincubated for 90 min with 1 μMof peptide. Chromium release was measured after 5 hours of incubation.^(c)gp100 280-288.

TABLE III HLA-A*0201 binding and complex stability of gp100154-162 epitope-analogues Affinity Stability peptide IC50 (μM)^(a)(DT 50%)^(b) FLPSDFFPSV^(C) (SEQ ID NO: 31) 0.5 >4 hrKTWGQYWQV (SEQ ID NO: 9) 1.4 >4 hr KTWGQYWAV (SEQ ID NO: 1) 0.5 >4 hrKVWGQYWQV (SEQ ID NO: 2) 0.8 >4 hr KTWGQYWQV (SEQ ID NO: 3) 0.4 >4 hrKIWGQYWQV (SEQ ID NO: 4) 0.6 >4 hr ^(a)Binding of peptides to HLA-A*0201was analyzed in a competition away at 4° C. using mild acid treatedHLA-A*0201⁺ B-LCL. The binding capacity of the peptides is shown as theconcentration of peptide needed to inhibit 50% of binding of theFluorescein labeled reference peptide. ^(b)The dissociation rate ofHLA-A*0201-peptide complexes was measured using emetine pretreatedHLA-A*0201⁺ B-LCL. After mild acid treatment, empty cell surfaceHLA-A*0201 molecules were loaded with peptide at room temperature andB-LCL were then put at 37° C. The decay of cell surface HLA-A*0201molecules was analyzed by flow cytometry. The dissociation rate isdepicted as the time required for 50% of the MHC class I-peptidecomplexes to decay at 37° C. ^(c)HBC 18-27, unlabeled reference peptide.

1-16. (canceled)
 17. A gp100 peptide consisting of SEQ ID NO:
 17. 18. Agp100 peptide consisting of SEQ ID NO:
 19. 19. A gp100 peptideconsisting of SEQ ID NO:
 21. 20. A gp100 peptide consisting of SEQ IDNO:
 23. 21. A gp100 peptide consisting of SEQ ID NO:
 32. 22. A gp100peptide consisting of SEQ ID NO:
 33. 23. A gp100 peptide consisting ofSEQ ID NO:
 34. 24. A pharmaceutical composition comprising the gp100peptide of claim
 17. 25. A pharmaceutical composition comprising thegp100 peptide of claim
 18. 26. A pharmaceutical composition comprisingthe gp100 peptide of claim
 19. 27. A pharmaceutical compositioncomprising the gp100 peptide of claim
 20. 28. A pharmaceuticalcomposition comprising the gp100 peptide of claim
 21. 29. Apharmaceutical composition comprising the gp100 peptide of claim
 22. 30.A pharmaceutical composition comprising the gp100 peptide of claim 23.31. Tumor infiltrating lymphocytes (TIL) which bind to the peptide ofclaim
 17. 32. A method of eliciting a T-cell response in a mammalcomprising administering to the mammal the peptide of claim 17 in anamount sufficient to elicit a T-cell response in a mammal.
 33. A methodof eliciting a T-cell response in a mammal comprising administering tothe mammal the peptide of claim 18 in an amount sufficient to elicit aT-cell response in a mammal.
 34. A method of eliciting a T-cell responsein a mammal comprising administering to the mammal the peptide of claim19 in an amount sufficient to elicit a T-cell response in a mammal. 35.A method of eliciting a T-cell response in a mammal comprisingadministering to the mammal the peptide of claim 20 in an amountsufficient to elicit a T-cell response in a mammal.
 36. A method ofeliciting a T-cell response in a mammal comprising administering to themammal the peptide of claim 21 in an amount sufficient to elicit aT-cell response in a mammal.